Giardia lamblia undergoes surface antigenic variation where only one of a family of structurally related proteins (variant-specific surface proteins, VSPs) is expressed on the surface at any one time. There are a number of conserved motifs in the family including one or two GGCY motifs, a Zn finger motif and a well conserved hydrophobic transmembranous portion followed by an absolutely conserved terminal 5 amino acids, CRGKA. Analysis of VSPs suggests that the amino terminal portion of VSPs is most diverse and antigenically distinct compared to the relatively well-conserved carboxyl terminal portion. However, why he hydrophobic membranous portion is so well conserved and why the terminal 5 amino acids are absolutely conserved is not known. The terminal amino acid is not required for proper insertion of VSP into the surface nor is the precise hydrophobic transmembrane sequence. However, conservation suggests these regions have important biological functions. Two posttranslational modifications were characterized. Previous studies by others showed that the VSPs are palmitoylated. Using a combination of transfection of VSP H7, HA and transfected into WB labeled with palmitate, we showed that VSPH7 was palmitoylated. Mutation studies revealed that palmitoylation occurred on the ?C? of CRGKA. WB Giardia expressing mutated non-palmitoylated VSPH7 as well as its own native VSPs appeared unchanged. However, palmitoylation changed the membrane domain and partitioning of VSPs suggesting that this may be a one biologically function. Experiments are planned to express both palmitoylated and non-palmitoylated in mammalian cells to determine if the palmitoylation permits partitioning into Rafts. The palmitoyl transferase responsible for VSP palmitoylation was identified and characterized. This transferase was able to transfer palmitoylate to in vitro translated VSPH7 and knockdown of this enzyme in vivo decreased palmitoylation of in vitro translated VSPH7. The second posttranslational modification that was discovered and characterized is the conversion of arginine in the CRGKA of VSPs to citrulline by arginine deiminase. Arginine deiminase in almost all prokaryotes and also Giardia functions in the conversion of free arginine to citrulline. Other studies in Giardia also showed it functioned similar to most bacterial deiminases. However, in mammals a peptidylarginine functions to convert protein bound arginine to citrulline and therefore is an enzyme is a post translation modification of VSPs. As mentioned above, the conserved nature of CRGKA VSP tail suggested a biological function and one way to determine the function was to use a poly-his CRGKA to pull down associated proteins. This technique yielded arginine deiminase. Two different VSPs tested both contained citrulline and as a more definitive proof purified recombinant Giardia arginine deiminase converted the arginine in the his-CRGKA peptide to citrulline. Furthermore, Giardia arginine deiminase localized to the plasma membrane and co localized with VSP as would be expected. Surprisingly, the enzyme localized to the nuclei during encystations. The biological function of the conversion to protein bound arginine to citrulline is not understood in most situations, but promises to be important in signaling and protein interactions.